The change in calcium ion distribution in cells is very important in the study of physiological and pathological phenomena. The calcium ion level in the cell is controlled by pumps or channels existing on the plasma membrane depending on various situations. The concentration of calcium ion is much higher near the cell membrane than its average value in the cell. Depending on physiological activity, it increases up to 100 μM or above. The highly concentrated calcium ions near the cell membrane are known to play important roles in exocytosis of hormones and neurotransmitters, as second messengers in signal transduction, or the like.
A typical mechanism of controlling the concentration of calcium ions is the Na+/Ca2+ exchanger (NCX). When the concentration of calcium ions in a cell increases, the NCX sends the calcium ions out of the cell in exchange for the import of sodium ions in order to maintain homeostasis. This process is called the Na+/Ca2+ exchange.
In order to study this phenomenon, a number of one-photon fluorescent probes have been developed. However, there is no case of imaging the two ions at the same time to study their interactions. Further, since most one-photon probes are problematic in that they have short extraction wavelengths (<500 nm), which limit application to tissue imaging because of shallow penetration depth (<100 μm), photobleaching and cellular autofluorescence.
An ideal solution to this problem is the two-photon microscopy (TPM) wherein two near infrared photons of low energy are used for excitation. The TPM allows a sustained imaging of intact tissue with minimum interference from tissue preparation artifacts that can extend more than 70 μm into the tissue slice. However, until now, there has not been developed a two-photon fluorescent probe capable of imaging the distribution of calcium ions near the cell membrane and sodium ions deep inside the living tissue (>100 μm) at the same time.